Diagnostic agent for detecting bilirubin

ABSTRACT

WHEREIN X is a stabilizing anion; R1 and R2, which may be the same or different, are halogen or lower alkyl or alkoxy and R3 and R4, which may be the same or different, are hydrogen, halogen, nitro, or lower alkyl or alkoxy; THE AROMATIC RING IN FORMULA (I) containing either (a) 1 to 3 halogen atoms or (b) one nitro group, but not both together; and an amount of an acid sufficient for a coupling reaction between bilirubin and said diazonium salt.   Bilirubin, particularly as contained in body fluids, is determined with test strips comprising an absorbent

United States Patent 1191 Rittersdorf et al.

1 Apr. 29, I975 1 DIAGNOSTIC AGENT FOR DETECTING BILIRUBIN [73]Assignee: Boehringer Mannheim G.m.b.II.,

Mannheim-Waldhof, Germany 221 Filed: July 24,1973

2| App1.No.:382,24l

[30] Foreign Application Priority Data Aug. 17, 1972 Germany 2240471[52] US. Cl. 23/253 TP; 23/230 B; 252/408 [51] Int. Cl G0ln 31/22; GOln33/16 [58] Field of Search....@ 23/230 B, 253 TP; 252/408; 8/41 D, 44

[56] References Cited UNITED STATES PATENTS 2,854,317 9/1958 Free at al.23/230 B 3,511,607 5/1970 Green 23/230 B 3,585,001 6/1971 Mast 1 23/230B 3,585,004 6/1971 Mast 23/230 B X 3,652,222 3/1972 Denny et a1. 23/230B 3,754,862 8/1973 Wahlefeld et al. 23/230 B Primary Examiner-Morris O.Wolk Assistant Examiner-Michael S. Marcus Attorney, Agent, orFirm--Burgess, Dinklage & Sprung 57 1 ABSTRACT Bilirubin, particularlyas contained in body fluids, is determined with test strips comprisingan absorbent wherein X is a stabilizing anion; R and R which may be thesame or different, are

halogen or lower alkyl or alkoxy and R and R which may be the same ordifferent, are hydrogen, halogen, nitro, or lower alkyl or alkoxy; thearomatic ring in formula (1) containing either (a) 1 to 3 halogen atomsor (b) one nitro group, but not both together; and an amount of an acidsufficient for a coupling reaction between bilirubin and said diazoniumsalt.

3I Claims, No Drawings DIAGNOSTIC AGENT FOR DETECTING BILIRUBIN Thepresent invention is concerned with an improved diagnostic agent for therapid and sensitive detection of bilirubin in body fluids, particularlyin urine.

The detection of bilirubin is of great importance in the diagnosis ofdiseased of the liver and gall bladder. Thus, in the case of liverdamage and of occlusion of the gall bladder duct, the early appearanceof bilirubin in the urine occurs even before the bilirubin content ofthe serum increases and clinical symptoms of jaundice appear. On theother hand, this type of jaundice can be differentiated from haemolyticicterus in which an increased bilirubin level can only be ascertained inthe serum but not in the urine.

Methods for the detection of bilirubin in the urine have been known foralong time. Of importance are the methods which depend upon theevaluation of the colored compounds formed by the coupling of bilirubinwith diazonium salts. For this purpose. diazosulfanilic acid haspreviously been most used.

Recently, in medical practice and in clinical laboratories, theso-called rapid tests have been used more and more as a diagnostic aid.These simple devices, usually test papers, which permit the carrying outof the detection or semi-quantitative determination of pathologicalcomponents of the body in a rapid and certain manner, even by untrainedpersonnel, such as medical auxiliaries.

Thus, for example, German Pat. No. 1,102,444 describes a reagent tabletcontaining a diazonium salt and a strong acid which is placed on a spotplate moistened with a body fluid and, after moistening with water,makes the bilirubin visible as a violet ring on the substrate.

It is obvious that such a diagnostic agent is relatively laborious touse. For a Wide use in medical practice and at the sick bed, it is,however, desirable that the rapid test is such that it can be read offwithout difficulty after simply dipping into the body fluid to beinvestigated. This purpose is best fulfilled by the so-called testpapers; these are absorbent carriers which have been impregnated withall the reagents necessary for the detection reaction.

It is known from German Pat. No. 2,007,013 to use test papers whichcontain certain 2,4- and 2,5- disubstituted diazonium salts for thedetection of bilirubin in serum and urine. However, this method isrelatively insensitive and, according to our investigations, alsosuffers from certain disadvantages with regard to the speed of thereaction and the sensitivity, which lessens its practical utility. Thereis, of course, the possibility of increasing the sensitivity of thedetection reaction but, in this case, new difficulties arise. Thus, theurobilinogen which is also present in urine also reacts in the presenceof these activators and simulates an increased biliburin content.Furthermore, the normal disturbing reactions with components of theurine are hereby also activated, which can manifest themselves as falsecolorations or even give falsely positive indications.

We have now, surprisingly, found that when using certain diazonium saltsas the detection reagents, the abovementioned difficulties no longeroccur, even when activators are used at the same time. Thus, thesediazonium salts can be used to prepare test strips which reactselectively with bilirubin not only in urine but also in serum.

The diazonium salts used in this invention are of the formula:

3 NEN (:9 X

wherein X is a stabilizing anion, R and R which may be the same ordifferent, are

halogen or lower alkyl or alkoxy and R and R,,, which may be the same ordifferent, are

hydrogen, halogen, nitro or lower alkyl or alkoxy; the aromatic ring informula (I) may contain either (a) l to 3 halogen atoms or (b) one nitrogroup, but not both together.

The diazonium salts of general formula (I) are either known or can beprepared from known aromatic amines by diazotization.

The lower alkyl or alkoxy radicals in the diazonium salts (I) are to beunderstood to contain up to 3 carbon atoms each.

The diazonium salts (I) are preferably used in the form of thefluoborates (i.e., wherein X is a fluoborate anion), the stability ofwhich is known; however, other stable salts, such as aryl-sulfonates,especially naphthalene-1,5-disulfonates, can also be used.

Diazonium salts of general formula (I) are preferred in which R and Rare both halogen atoms, especially chlorine or bromine atoms, and theother symbols stand for hydrogen atoms.

As activators, there can be used, apart from anionic wetting agents,especially compounds of the general formula:

. wherein R and R which may be the same or different, are unsubstitutedor substituted aliphatic, cycloaliphatic, araliphatic or aromaticradicals.

The radicals R and R are preferably identical since phosphoric aciddiesters of this type are particularly simple to prepare. The aliphaticradicals R and R can be straightchained or branched and can contain upto 18 carbon atoms, the effectiveness of the esters initially increasingwith an increasing number of carbon atoms but beyond a chain length ofabout 14 carbon atoms, the effectiveness again decreases due to theincreasingly hydrophobic character of the esters. As cycloalkylradicals, those with 5-8 carbon atoms are especially preferred.Preferred substituents in the aliphatic and cycloaliphatic radicalsinclude, for example, halogen atoms, especially chlorine and bromineatoms, nitro groups and alkoxy radicals containing up to 8 carbon atoms.With regard to the stability of the test strips, phosphoric aciddiesters of general formula (II) are preferred in which R and R bothrepresent aromatic radicals. Preferred aromatic radicals include monoandpolynuclear, unsubstituted and substituted aryl radicals, for example,phenyl, xylyl, tolyl, chlorophenyl, nitrophenyl and naphthyl radicals.As araliphatic radicals, there are preferably used, for example, phenylor naphthyl radicals which are connected to the phosphoric acid residuevia an alkylene bridge containing up to 3 carbon atoms.

For the preparation of an especially useful test strip according to thepresent invention, an absorbent carrier, preferably filter paper, isimpregnated with a solution which contains 0.0 l-2.0 g., preferably0.050.5 g., per 100 ml. of a diazonium salt of general formula (I), aswell as 3-20 g., preferably 5-15 g., per 100 ml. of a phosphoric aciddiester of general formula (II) and 230 g. of a solid inorganic ororganic acid and thereafter dried. However, it is also possible to carryout the v diazotization in the impregnation solution or on the absorbentcarrier. In this case, an appropriate amine is used as starting materialwhich is then reacted with a nitrite in the usual manner.

The bilirubin test strips according to the present invention can alsocontain stabilizing agents for the diazonium salts, for example sodiumfluoborate, magnesium sulfate, sodium metaphosphate, aryl-sulfonates orthe like.

Furthermore, the test strips can also contain wetting agents in order toimprove the absorbency of the test papers. In principle, all wettingagents can be used which are still surface-active in the strongly acidicmedium after dipping into a body fluid to be tested, for examplecationic agents (e.g. lauryl-pyridinium chloride), non-ionic agents(e.g. polyoxyethylene triglyceride) and anionic agents, especially thesulfonates and sulfates (e.g. dodecyl-benzene-sulfonate). The latter canadditionally possess activating properties.

The wetting agents can be used in the impregnation solution inconcentrations of O. l2% and preferably of 0.2-0.5%.

As solvents or solvent mixtures for the impregnation, there can be usedall those which do not react with the diazonium salts, in which all thecomponents are soluble and which have a low boiling point in order that,upon drying, the diazonium salts are not subjected to too high atemperature. It has also proved to be advantageous to impregnateindividual components in separated working steps. Thus, for example, amixture of a diazonium salt (I) and an acid, for example metaphosphoricacid, can first be used for impregnation from an aqueous solution andthen a further impregnation is carried out with a solution of aphosphoric acid diester (II) in ethyl acetate or chloroform.

As absorbent carrier, it is preferred to use filter paper but otherabsorbent carriers, for example, glass fiber paper and synthetic fiberfabrics and fleeces made from acid-resistant fibers, such as polyesterand polypropylene fibers, can also be used. The term test paper usedherein is to be understood to include these materials.

Thus, the present invention provides a test strip for the detection ofbilirubin in body fluids, which comprises an absorbent carriercontaining at least one diazonium salt of general formula (I) and anamount of an acid sufficient for the coupling reaction.

Phosphoric acids of general formula (II) are known (see, for example,l-Iouben-Weyl, Methoden d. Org. Chem., Vol. XII/2, pp 226 et seq.).Since the compounds (II) themselves have an acidic reaction, they can atleast partially replace the acid needed for the coupling reaction.However, additional amounts of acid are usually necessary. For thispurpose, a whole series of solid acids can be used. Of particular useare, for example, oxalic acid, citric acid, potassium bisulfate and,especially having regard to the stability of the diazonium salt,commercially available metaphosphoric acid, which can contain up toabout of its sodium salt.

The test strips according to the present invention react withbilirubin-containing urine in about 5 to 30 seconds to give red to bluecolor shades. With normal urine, there is obtained a yellow reactioncolor. Urobilinogen reacts, without disturbing, with a yellow color, redshades only occurring when unusually large amounts thereof are presentin the urine (more than about 8-10 mg.%). The detection of serumbilirubin is, of course, also possible with the use of the test stripsaccording to the present invention.

The impregnated absorbent carriers can be cut up into long strips andthen rolled up so that, when needed, a small piece can be torn or cutoff. They can also be cut up into small rectangles and stuck or sealedon to one end of narrow strips of synthetic resins. It is of particularadvantage when the test papers are sealed between two synthetic resinfilms according to the method described in German Patent No. l,546,307or between a synthetic resin film and a meshwork as described in GermanPatent No. 2,1 18,455, because there is then no danger that the reagentswill be washed out upon dipping into a body fluid.

The following Examples are given for the purpose of illustrating withoutlimitation, the present invention:

EXAMPLE 1 Filter paper was successively impregnated with the followingsolutions and then dried at 40C.

Solution 1:

2,o-dichlorobenzene-diazonium fluoborate 0.08 g. metaphosphoric acidl0.0 g. citric acid 3.0 g. distilled water ad 100.0 ml. Solution II:

phosphoric acid diphcnyl ester 5.0 g dodecyl-benzenc-sulfonic acid:

sodium salt 0.4 g. ethyl acetate ad 100.0 ml.

With bilirubin-containing serum, there were obtained red-violetcolorations of increasing intensity, the limit of sensitivity beingabout 0.4 to 0.6 mg.%.

Test papers which contain 2,6-dib'romobenzenediazonium fluoboratereacted in an analogous manner.

Test papers of analogous composition but which con tain2,4-dichlorobenzene-diazonium fluoborate (of. German Pat. No. 2,007,013)reacted with bilirubin with a blue color. With urobilinogen-containingurine,

they gave a deep violet coloration after only a few seconds. 1

EXAMPLE 2 Test papers which reacted in a manner analogous to thatdescribed in Example 1 were obtained when the phosphoric acid diphenylester was replaced by one of the following phosphoric acid esters:

phosphoric acid di-o-tolyl-ester; phosphoric acid di-p-tolyl ester;phosphoric acid bis-3,5-xylol ester; phosphoric acid bis-o-chlorophenylester; phosphoric acid bis-p-chlorophenyl ester; phosphoric acidbis-p-nitrophenyl ester; phosphoric acid dicyclohexyl ester; orphosphoric acid dipentyl ester.

EXAMPLE 3 Filter paper was impregnated with a solution of the followingcompoisition and then dried at 40C.:

2,6-dichlorobcnzcne-diazonium fluoborate 0.16 g. metaphosphoric acid15.0 g. dodecyl-benzene-sulfonic acid sodium salt 1.0 g. methanol 20.0ml. distilled water ad 100.0 ml.

This test paper reacted, after 20-30 seconds, with a violet color withbilirubin-containing urine, the sensi tivity limit being about 0.7 to0.9 mg.%. With urine having a high content of urobilinogen (about 8-12mg.%), a pale red color appeared after about 1-2 minutes.

A test paper of the same composition except that it contained2,4-dichlorobenzene-diazonium fluoborate, reacted with bilirubin with ablue-violet color and, with urobilinogen-containing urine, a disturbingviolet coloration occurred after only a short period of time.

EXAMPLE 4 Filter paper was impregnated with a solution of the followingcomposition and then dried at 40C.:

2-mcthoxy-4,6-dibromobcnzcne-diazonium fluoborate 0.12 g. citric acid10.0 g. sodium mctaphosphatc 3.0 g. distilled water ad 100.0 m1.

This paper was then impregnated with a 5% solution of phosphoric aciddiphenyl ester in ethyl acetate.

The properties of this test paper, as well as of test papers containingan equimolar amount of one of the diazonium salts set out in thefollowing Table l, corresponded to those of Example 1. The reactioncolors with bilirubin were, in the case of the halogenandalkyl-substituted compounds, more blue-violet and, in

the case of the alkoxyand nitro-substituted compounds, more grey-blue.

TABLE I EXAMPLE 5 A solution of the following composition was preparedat 510C., then impregnated on to filter paper and dried at 40C.:

2-methy1-6-chloroaniline 0.14 g sodium nitrite 0.07 g metaphosphoricacid 10.0 g distilled water ad 100.0 ml

This test paper was thereafter impregnated with a 10% solution ofphosphoric acid diphenyl ester in ethyl acetate and dried at 40C.

Analogous test papers could be prepared with an equimolar amount of oneof the following substituted aniline derivatives:2,6-dimethyl-3-bromoaniline; 2,6-dimethyl-4-bromoanilinei2,6-dimethoxy-3- bromoaniline; and 2,6-dimethoxy-4-chloroaniline.

The properties of the test papers obtained correspond approximately tothose of the test papers according to Examples 1 and 4.

EXAMPLE 6 Filter paper was impregnated with a solution of the followingcomposition and dried at 40C.:

2,4,6-trichlorobenzene-diazonium toluenesulfonate 0.3 g. metaphosphoricacid 10.0 g. distilled water ad 100.0 ml.

This paper was subsequently impregnated with a 10% solution ofcommercially available isooctyl-phosphoric, acid in methylene chloride.This acid is a mixture of about 55% of the diester and 45% of themonoester of phosphoric acid with isomeric C alcohols.

The properties of this test paper corresponded to those of the testpaper according to Example 1.

Test papers which contain an equimolar amount of one of the followingdiazonium salts possess practically the same properties:

2,4,6-tribromobenzene-diazonium naphthalenedisulfonate;2,4,6-tribromo-3-methyl-benzene-diazonium fluoborate;

2-methyl-4,6-dibromo-S-chlorobenzene-diazonium fluoborate;

2,4,6-tribromo-3-methoxy-benzene-diazonium fluoborate;

2,3,6-tribromobenzene-diazonium fluoborate.

wherein X is a stabilizing anion,

R, and R which may be the same or different, are

halogen or lower alkyl or alkoxy and R and R.,, which may be the same ordifferent, are

hydrogen, halogen, nitro, or lower alkyl or alkoxy; the aromatic ring informula (1) containing either (a) l to 3 halogen atoms or (b) one nitrogroup, but not both together; and an amount of an acid sufficient for acoupling reaction between bilirubin and said diazonium salt.

2. Test strip as claimed in claim 1, wherein R, is halogen.

3. Test strip as claimed in claim 1, wherein R is alkyl of from I to 3carbon atoms.

4. Test strip as claimed in claim 1, wherein R, is alkoxy of from 1 to 3carbon atoms.

5. Test strip as claimed in claim 1, wherein R is halogen.

6. Test strip as claimed in claim 1, wherein R is alkyl of from 1 to 3carbon atoms.

7. Test strip as claimed in claim 1, wherein R is all;- oxy of from 1 to3 carbon atoms.

8. Test strip as claimed in claim 1, wherein R is hydrogen.

9. Test strip as claimed in claim 1, wherein R is halo gen.

10. Test strip as claimed in claim 1, wherein R is nitro.

11. Test strip as claimed in claim 1, wherein R is alkyl of from 1 to 3carbon atoms.

12. Test strip as claimed in claim 1, wherein R is alkoxy of from I to 3carbon atoms.

13. Test strip as claimed in claim 1, wherein the aromatic ring of saiddiazonium salt contains a nitro group and no halogen atoms.

14. Test strip as claimed in claim 1, wherein the aromatic ring of saiddiazonium salt contains one halogen atom and no nitro group.

15. Test strip as claimed in claim 1, wherein the aromatic ring of saiddiazonium salt contains two halogen atoms and no. nitro group.

16. Test strip as claimed in claim 1, wherein the aromatic ring of saiddiazonium salt contains three halogen atoms and no nitro group.

17. Test strip as claimed in claim 1, wherein said dia-- zonium salt is2,6-dichlorobenzene-diazonium fluoborate.

7 18. Test strip as claimed in claim 1, wherein said diazonium salt is2-methoxy-4,6-dibromobenzenediazonium fluoborate.

19. Test strip as claimed in claim 1, wherein said diazonium salt is2,4,6-trichlorobenzene-diazonium toluenesulfonate.

20. Test strip as claimed in claim 1 also containing an activator.

21. Test strip as claimed in claim 20, wherein said activator is aphosphoric acid diester of the general formula:

in which R and R,,, which may be the same or different, areunsubstituted or substituted aliphatic, cycloaliphatic, araliphatic oraromatic radicals.

22. Test strip as claimed in claim 1, wherein the stabilizing anion X isa fluoborate or aryl-sulfonate ion.

23. Test strip as claimed in claim 1, wherein the absorbent carrier isimpregnated with a solution containing, per ml., 0.0l2.0 g. of thediazonium salt, 3-20 g. of the phosphoric acid diester and 2-30 g. of asolid inorganic or organic acid.

24. Test strip as claimed in claim 1, wherein the absorbent carrier isimpregnated with a solution containing 0.050.5 g. of the diazonium saltper 100 ml.

25. Test strip as claimed in claim 1, also containing a stabilizingagent.

26. Text strip as claimed in claim 25, wherein the stabilizing agent issodium fluoborate, magnesium sulfate, sodium metaphosphate or anaryl-sulfonate.

27. Test strip as claimed in claim 1,.wherein said absorbent carrier isa filter paper, glass fiber paper or a fabric or fleece made fromacid-resistant synthetic fibers.

28. Test strip as claimed in claim 1, wherein said absorbent carrier isadhered to one end of a narrow strip of synthetic resin.

29. Test strip as claimed in claim 1, wherein said absorbent carrier issealed between two synthetic resin films.

30. Test strip as claimed in claim 1, wherein said absorbent carrier issealed between a synthetic resin film and a meshwork.

31. Method for detecting bilirubin in body fluids, which methodcomprises contacting a test sample suspected of containing bilirubinwith a test strip as claimed in claim 1.

UNITED STATES PATbNl OIFICE CERTIFICATE OF CORRECTION I at 3.880.588Dated April 29 1975 Q 1 Walter Rittersdorf et al It is certified thaterror appears in the aboveidentified patent I and that said LettersPatent are hereby corrected as shown below:

Under the heading ABSTRACT at line 2 after the word "absorbent" insert--carrier containing a diazonium salt of the formula:-. Q

Column 1, line 8, delete "diseased" and substitute therefor --diseases-.

Column 4, line 54, after the word "acid" delete --the colon Column 5,line 31, delete "compoisition" and substitute therefor --composition-.

Signed and Ewaled this 0 second Day Of September 1975 [SEAL] Arrest:

Q RUTH C. MASON c. MARSHALL DANN Arlvsling Officer (mnmissimwr uflatemsand Trademarks FORM PO-IOSO (10-69) USCOMM-DC 60316-P59

1. TEST STRIP FOR THE DETECTION OF BILIRUBIN IN BODY FLUIDS, WHICHCOMPRISES AN ABSORBENT CARRIER CONTAINING A DIAZONIUM SALT OF THEFORMULA:
 2. Test strip as claimed in claim 1, wherein R1 is halogen. 3.Test strip as claimed in claim 1, wherein R1 is alkyl of from 1 to 3carbon atoms.
 4. Test strip as claimed in claim 1, wherein R1 is alkoxyof from 1 to 3 carbon atoms.
 5. Test strip as claimed in claim 1,wherein R2 is halogen.
 6. Test strip as claimed in claim 1, wherein R2is alkyl of from 1 to 3 carbon atoms.
 7. Test strip as claimed in claim1, wherein R2 is alkoxy of from 1 to 3 carbon atoms.
 8. Test strip asclaimed in claim 1, wherein R3 is hydrogen.
 9. Test strip as claimed inclaim 1, wherein R3 is halogen.
 10. Test strip as claimed in claim 1,wherein R3 is nitro.
 11. Test strip as claimed in claim 1, wherein R3 isalkyl of from 1 to 3 carbon atoms.
 12. Test strip as claimed in claim 1,wherein R3 is alkoxy of from 1 to 3 carbon atoms.
 13. Test strip asclaimed in claim 1, wherein the aromatic ring of said diazonium saltcontains a nitro group and no halogen atoms.
 14. Test strip as claimedin claim 1, wherein the aromatic ring of said diazonium salt containsone halogen atom and no nitro group.
 15. Test strip as claimed in claim1, wherein the aromatic ring of said diazonium salt contains two halogenatoms and no nitro group.
 16. Test strip as claimed in claim 1, whereinthe aromatic ring of said diazonium salt contains three halogen atomsand no nitro group.
 17. Test strip as claimed in claim 1, wherein saiddiazonium salt is 2,6-dichlorobenzene-diazonium fluoborate.
 18. Teststrip as claimed in claim 1, wherein said diazonium salt is2-methoxy-4,6-dibromobenzene-diazonium fluoborate.
 19. Test strip asclaimed in claim 1, wherein said diazonium salt is2,4,6-trichlorobenzene-diazonium toluenesulfonate.
 20. Test strip asclaimed in claim 1 also containing an activator.
 21. Test strip asclaimed in claim 20, wherein said activator is a phosphoric acid diesterof the general formula:
 22. Test strip as claimed in claim 1, whereinthe stabilizing anion X is a fluoborate or aryl-sulfonate ion.
 23. Teststrip as claimed in claim 1, wherein the absorbent carrier isimpregnated with a solution containing, per 100 ml., 0.01-2.0 g. of thediazonium salt, 3-20 g. of the phosphoric acid diester and 2-30 g. of asolid inorganic or organic acid.
 24. Test strip as claimed in claim 1,wherein the absorbent carrier is impregnated with a solution containing0.05-0.5 g. of the diazonium salt per 100 ml.
 25. Test strip as claimedin claim 1, also containing a stabilizing agent.
 26. Text strip asclaimed in claim 25, wherein the stabilizing agent is sodium fluoborate,magnesium sulfate, sodium metaphosphate or an aryl-sulfonate.
 27. Teststrip as claimed in claim 1, wherein said absorbent carrier is a filterpaper, glass fiber paper or a fabric or fleece made from acid-resistantsynthetic fibers.
 28. Test strip as claimed in claim 1, wherein saidabsorbent carrier is adhered to one end of a narrow strip of syntheticresin.
 29. Test strip as claimed in claim 1, wherein said absorbentcarrier is sealed between two synthetic resin films.
 30. Test strip asclaimed in claim 1, wherein said absorbent carrier is sealed between asynthetic resin film and a meshwork.
 31. Method for detecting bilirubinin body fluids, which method comprises contacting a test samplesuspected of containing bilirubin with a test strip as claimed in claim1.